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September | October 2011

Feature title: Partial replacement of artemia:Evaluation of formulated larval diets for giant freshwater prawn macrobrachium rosenbergii

The International magazine for the aquaculture feed industry

F: Larval diets

The giant freshwater prawn (Macrobrachium rosenbergii, de Man), locally known as ‘Golda’, plays an important

role in the economy of Bangladesh by earning valuable foreign exchange, generating employment and raising protein food production (Chowdhury et al., 2010).

Being a protein diet food and also for its big size, it is increasingly attracting the attention of the culturists as well as consumers.

Freshwater prawn farming is environ-mentally sustainable, since it is practiced at lower grow-out density (New, 1995). Adults breed very readily during summer wet season and in captivity, however, the larvae develop through 11 stages, requiring a moderate level of hatchery technology and considerable husbandry skill for rearing (New and Singholka, 1982).

The success of this system depends on its proper operation and management.

Good feeding regime is important for the optimum production in hatchery condi-tion. Quality feed plays an important role in larvae rearing of freshwater prawn. It affects

survival, growth, disease and larval qual-ity as well. Under or over feeding regime causes economic losses. Proper feeding with optimum ration size is key factor in larval rearing of Macrobrachium rosenbergii.

It helps to create maximum economic efficiency, healthy and disease resistant post larvae (PL) and finally excellent aquaculture products. So, the selection of best larval feeding regime of cultured Macrobrachium rosenbergii in the larval rearing tanks should be considered more carefully for the supreme success of the hatchery.

Imported Artemia cysts are predomi-nantly used in the hatcheries of Bangladesh, which are expensive and uncertain in avail-ability. Dependence entirely on Artemia as feed not only makes hatchery operations expensive, but also unsustainable.

Hence, there is a need to look for alter-native acceptable diets to replace Artemia and reduce the cost of larval prawn rearing.

Accordingly, an attempt was made in the present study to find out the growth, surviv-al rate and disease stress of Macrobrachium larvae under different feeding regimes to replace Artemia partially in freshwater prawn hatchery.

MethodologyThree larval diets were formulated using

different feed ingredients (see Table 1). Feed ingredients were added and

blended thoroughly in a kitchen mixer. The diets were cooked on a water bath for 30 minutes, cooled and stored at 4°C. Six cemented rectangular tanks having 3m3 capacity (1.5mX2mX1m) were used for the experiment.

Three treatments (T1, T2 and T3) were arranged with one replicate for each treatment. 150‰ brine were collected from the near by sea salt extraction pen and stored.

To get 12‰ saline water, required quan-tities of brine was mixed with freshwater.

The larvae were stocked 100inds/L with 45cm UV treated 12ppt saline water at first maintaining all physicochemical parameters

following the FAO manual (New, 2002) and then, increased the water level up to 90cm as the larvae grew (see Figure 1a).

The larval feeding regime used was as described in Table 2.

Diets were offered to the prawn larvae three times daily (09:30, 01.30 and 03:30 hours) at a rate of 5-20g/tonne (see Figure 1b).

Three larval diets were formulated and evaluated in partial replacement for Artemia in the larval rearing of Macrobrachium rosenbergii.

The research work was conducted using six cemented rectangular tanks having 3m3 capacity (1.5mX2mX1m) each. 100 larvae per liter were stocked in three experimental setups as the treatments T1, T2 and T3 respectively with one replicate each.

The test diets were prepared by using egg custard (T1), egg-shrimp custard (T2), and formulated egg-shrimp custard (T3). Diets were offered to the prawn larvae three times daily (09:30, 01.30 and 03:30 hrs) at a rate of 5-20g/tonne.

Formulated egg-shrimp custard (T3) showed significantly higher survival than those fed egg custard (T1) and egg-shrimp custard (T2). The larvae fed formulated egg-shrimp custard (T3) was more efficient in larval metamorphosis, larvae took less time to reach the next stage compared to those fed egg custard (T1) and egg-shrimp custard (T2).

Length and weight measurements were higher for the larvae fed formu-lated egg-shrimp custard (T3) and the formulated egg-shrimp custard provided the necessary nutrition to reduce the risk of larval exuvia entrapment disease.

Water quality viz., temperature, pH, dissolved oxygen (DO), salinity, alkalinity, nitrite- nitrogen (NO2- N) and ammonia (NH3) were not affected by the test diets and the values recorded were in acceptable range for freshwater prawn larval rearing.

Partial replacement of artemia: Evaluation of formulated larval diets for giant freshwater prawn macrobrachium rosenbergii

by M Shah Nawaz Chowdhury, Institute of Marine Sciences and Fisheries, University of Chittagong,

Chittagong 4000, Bangladesh Email: snawaz11@yahoo.com

Table 1: the composition of the test diets

test diets

Ingredients (%)t1

(eC)t2

(eFC)t3

(FeSC)

Eggs 50.00 50.00 31.00Skim milk powder - - 29.50

Shrimp muscle - - 26.50Fish meat - 30.00 -

Corn Flower 49.00 19.00 08.50Cod liver oil - - 02.00

Agar powder - - 01.00Vitamin C - - 00.20

Other multivitamins - - 00.50Yeast 01.00 01.00 00.60

Mannan-oligosaccharide (MOS) - - 00.10

Betaglucan - - 00.10

12 | InternatIOnal AquAFeed | September-October 2011 September-October 2011 | InternatIOnal AquAFeed | 13

Before feeding, the diets were passed through desired mesh size sieves (250-1000μ), so that the particle size of the food was acceptable to the prawn larvae. Live Artemia nauplii were fed twice daily at 07:30 hours and 6:30 at a rate of 1-5 individuals/ml of tank water.

Biochemical analysis of newly hatched Artemia and formulated diets was car-ried out (see Table 3) following standard methods of AOAC (1995). Siphoning has been done daily afternoon for removal of waste particles.

To maintain an optimum environment for larval growth, 20-30 percent volume water exchange has been done every day. The water removed has been replaced by ready mix, aerated, 12ppt salinity of water at the same temperature as that of the larval tank.

Water quality was monitored by observ-ing the physicochemical parameters viz. water temperature, water pH, Dissolved Oxygen (DO), Salinity, and Alkalinity, Nitrite- nitrogen (NO2- N) and Ammonia (NH3) (see Figure 1c).

Survival rate was measured by the volu-metric estimation of stocked larvae in each tank (see Figure 1d). The larval behavior

was visually checked twice a day after feeding and early morning to determine the larval conditions that is, swimming pattern, pigmentation, and accumulation in the tank bottom after stopped aeration.

Besides, significant number of larvae were sampled daily from each treatment tank for microscopic observation to deter-mine the mean larval stage and diagnose the disease and following symptoms described by FAO manual (New, 2002).

The growth was measured with cen-timeter scale for length (from tip of the rostrum to the end of the telson) and weighing by electrical balance.

Efficiency of experimental diets was evaluated on the basis of postlarval growth, time required for metamorphosis and related percentage survival. The larvae were observed daily to record the progression of metamorphosis namely, mean larval stage (MLS) and relative percentage survival (see Figure 1e, and 1f).

Larvae from each tank were collected and studied for morphological charac-ters following the descriptions given by Uno and Soo (1969). Development of larvae was determined by calculating the MLS formulas described by Lovett

Fig. 1 a. Larvae stocking in the larval rearing tank (LRT); b. Larval feeding of formulated diets in LRT; c. Water quality monitoring in the experimental LRTs; d. Larvae sampling for survival rate estimation; e. Microscopic observation of larvae (early stage); f. Microscopic observation of larvae (later stage); g. Harvested post larvae (PL) during this trial

12 | InternatIOnal AquAFeed | September-October 2011 September-October 2011 | InternatIOnal AquAFeed | 13

F: Larval diets

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The overall growth rate of larvae in treatment T3 was faster than T1, and T2. Similar result was observed in the rates of metamorphosis.

Survival rateFigure 4 shows

Formulated feed had (T3) enhanced

the rate of metamorphosis. The mean larval stages were dissimilar

among T1, T2 and T3. The first postlarvae were observed after 26, 24, and 22 days of rearing in T1, T2 and T3 respectively.

When the postlarval stage appeared, it was easier to identify by the change of swimming to crawling on the bottom or the wall of the tank.

However, 100 percent larvae (survived) were metamorphosed at the 41st, 38th and 35th day for treatment T1, T2 and T3 respectively.

At the end of rearing cycle the survival rate of larvae were found 22.8 percent, 31.6 percent and 38.7 percent in treatments T1, T2 and T3 respectively. So, the survival rate was comparatively higher in T3 than T2 and T1.

DiseaseSevere Zoothanium infestation was

observed in all treatments, which colonized in the culture media and exoskeleton of larvae causing mortality in these trials (see Figure 5a, and 5b). Then to improve the water quality 30 percent water has been changed. EED was observed in late stage (X-PL1) larvae and early post larvae in the T1 where the highest number larvae were initially stocked (see Figure 5c). The cause of EED is unknown but it is thought that nutritional deficiency may be the limiting factor.

The tanks in which larvae were offered formulated egg-shrimp custard (T3) with Artemia had higher survival rate (38.7%) than larvae offered egg custard (T1) (22.8%) and egg prawn custard (T2) (31.6%).

The larvae fed formulated egg-shrimp custard (T3) took less time to reach the next stage than that egg custard (T1) and egg shrimp custard (T2) diets, which indi-cates efficiency of formulated egg custard diet.

Higher survival, MLS, growth and meta-morphosis were recorded in the T3, prob-ably attributed to the diet which contained different ingredients which are known

phosed to post larvae, the experiment was terminated.

All the post larvae were harvested from each tank and counted (see Figure 1g). Two-way analysis of Variance and Duncan’s multiple range tests were used to test the significant difference among the treatments (Snedecor and Cochren 1968, Duncan 1955).

Water quality During the study period, water tempera-

ture in different larval rearing tanks were ranged from 28.5-30.5°C in the morning and 29.0-31.5°C in the afternoon.

The mean water temperatures were recorded as 30.1°C, 30.2°C, and 30.1°C in T1, T2 and T3 respectively. Water pH values were ranged (7.5-8.2) in the morning and (7.8-8.2) in the evening. The mean DO val-ues were recorded 5.8 mg/L, 5.8 mg/L, and 5.9 mg/L in different treatments T1, T2 and T3 respectively. The mean values of NO2-N concentration were found 0.19 mg/L, 0.18 mg/L, and 0.15 mg/L in treatments T1, T2 and T3 respectively.

The mean values of NH3 concentration were found 0.29 mg/L, 0.26 mg/L, and 0.23 mg/ in treatments T1, T2 and T3 respec-tively. The mean values of alkalinity were recorded 145 mg/L, 147 mg/L, and 145 mg/L in T1, T2 and T3 respectively.

Growth rateGrowth rate in terms of body length was

0.127mm/day, 0.135mm/day, and 0.168mm/day in T1, T2 and T3 respectively whereas growth rate in terms of body weight was observed 0.000115g/day, 0.000180g/day, and 0.000240g/day in T1, T2 and T3 respectively (see Figure 2 and Figure 3).

From the beginning of rearing (stage I) until the stage VI there was no significant difference in the growth rate of larvae (0.3mm/day) in each treatment.

In stage VII and VIII faster (1.017mm/day) growth was observed whereas in stage IX to XI moderate (0.367mm/day) growth was recorded.

and Felder (1988): MLS = ∑(Si*Ps), where, Si is the larval stage number (i=1,2,3,…11) and Ps is the proportion of larvae at that stage.

All materials used for each experimental unit were separated and disinfected by using iodine solution to avoid any cross contamination. When more than 95 percent of the larvae in all the tanks had metamor-

Table 2: Feeding regimes used for the treatments t1, t2, and t3 in larval rearing

Treat-ments

Feed Items

Feeding times

Larval stages

I II III IV V VI VII VIII IX X XI PL1

T1, T2, T3

Artemia nauplii

07.3018.30

4-5 nauplii/ml 2-3 nauplii/ml

1-2 nauplii/ml

T1 EC09.3001.3003.30

5-10 g/ton 10-20 g/ton

T2 EFC09.3001.3003.30

5-10 g/ton 10-20 g/ton

T3 FESC09.3001.3003.30

5-10 g/ton 10-20 g/ton

Fig. 2 Larval growth (length) in 3 different treatments

Fig. 3 Larval growth (weight) in 3 different treatments

Fig. 4 Larval development (days) in three different treatments

14 | InternatIOnal AquAFeed | September-October 2011 September-October 2011 | InternatIOnal AquAFeed | 1514 | InternatIOnal AquAFeed | September-October 2011 September-October 2011 | InternatIOnal AquAFeed | 15

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mulated egg-shrimp custard) with optimum water quality management in larval rearing, EED could prevent.

ConclusionBy incorporating meat of shrimp, chicken

eggs, skimmed milk powder, corn flowers, agar powder, cod liver oil and other neces-sary vitamins in formulated diets, enhanced growth, faster metamorphosis and survival of prawn larvae when fed in combination with Artemia than other common diets, which were widely used in prawn hatcher-ies in Bangladesh.

This formulated diet could effectively use to replace the use of Artemia partially in the larval rearing of prawn, which can provide all nutrition for prawn larval growth and reduce the production cost.

ReferencesAvailable on request

requires more than 4mg/l of dissolved oxygen (Reddy, 1997).

NH3 is the principal excretory metabol-ic of prawns and is generally considered a major cause of death in hatchery condition.

In a conditioned system, a bacterial nitri-fication process converts NH3 to relative non-toxic NO3 where as the intermediate product NO2 is highly toxic to aquatic vertebrates. Observed NO2- N concentra-tion was ranged from 0.1-0.3mg/l where the Macrobrachium larvae can tolerate 1.8mg/l concentration (Amstrong et al., 1976). Malecha (1980) reported that survival was nearly 50 percent in hatcheries, but less than one percent in the wild.

In the present trial, 21.9 percent, 31.0 percent and 43.0 percent survival rate was achieved in treatments T1, T2 and T3 respec-tively under different feed regimes. During late larval stages, mortality can occur due to cannibalism (Suharto et al., 1980).

Similar to that report, in the present trail cannibalism was observed when the larvae metamorphosed into post larvae immediately after larval molting. The mor-tality caused by cannibalism was high when the larval population was well dispersion or was insufficient nutrition or both. Mortality of larvae was also observed as larvae jumped and got stranded on tank wall above the water surface.

Larvae were seen to start jumping after stage VIII, especially after feeding with pre-pared feed. To reduce this mortality, aeration was adjusted and the stranded larvae on the tank wall were more frequently rinsed.

In addition, the mortality caused by disease was the most serious case. Though, different diseases viz., protozoan infestation, EED were diagnosed in this trail, but the Zoothanium had the severe effects on larvae than other occurred diseases and their presence was observed in the water and exoskeleton of larvae causing mortal-ity in the all treatments, Zoothanium was found all stage of the larval development. T1 resulted in mass mortality due to the Zoothanium, EED diseases.

By this investigation, it was observed that EED affects late stage larvae and early post larvae where the affected larvae were unable to free appendages, eyes or rostrum from the exuvia in which they became entrapped.

Other larvae, which shed the exuvia, have malformed appendages and die shortly after molting. This observation was com-pletely coincided with Brock (1983, 1988).

Another important finding was that EED occurred in T1, T2 and there was no pres-ence of EED in T3, which reveals that by providing well formulated larval feed (for-

to contain higher HUFAs and necessary vitamins.

Cod liver oil contains ω3 HUFA’s, which are important for prawn larval growth and survival (Murthy, 1998). Formulated diet that contained varying levels of chicken egg, milk powder and shrimp muscle in combi-nation with Artemia yielded better growth and survival of prawn larvae (Rao, 1997).

Artemia nauplii are the predominant live food used for larval rearing in prawn hatcheries.

Though supplementation of Artemia with prepared feed in prawn larval rearing have been reported (Sick and Beaty 1975, Corbin et al. 1983), no standard substitute for Artemia has been developed in freshwater prawn hatcheries. In view of the high cost of cysts and their occasional scarcity, too much dependence on Artemia is a major constraint

in the expansion of Macrobrachium rosenbergii hatcheries (New 1990).

These are some of demerits of Artemia in addition to its cost and availability factors. Bacterial degradation of larval exuvia and empty shells of Artemia foul the water, accumulated debris entangles larvae and leads to mortality.

The cysts which are ingested by the lar-vae cannot be digested and may cause block-age of the gut and have other deleterious effects (Stults, 1974). There is considerable variation in nutritive quality (Particularly HUFAs) of Artemia cysts among different geographical strains, (Murthy 1998).

During this study, the water tempera-ture were ranged from 28.5°C to 31.5°C. New and Singholka (1985) reported the optimal temperature in prawn larval rearing water which could range from 26-31°C. The research was designed to maintain the standard salinity 12‰ for larval rearing in all treatments until complete metamorphosis, which was strongly recommended (New and Singholka, 1985; Ling, 1969; Fijimura, 1974).

In the present study, the pH was ranged from 7.5 to 8.2 where the various authors suggested 7.0 to 8.5 for prawn larval rear-ing (Ling 1969, Aquacop 1983, and New, 1990). The average dissolved oxygen level varied from 5.8 to 5.9mg/l. Prawn larvae

Table 3: Proximate composition of the two test diets and artemia nauplii (% dry weight ± SD)

Diets Protein lipid ash

T1 (EC) 44.03±0.80 7.83±0.35 1.96±0.03T2 (ESC) 47.11±0.89 8.21±0.27 2.79±0.05

T3 (FESC) 51.03±0.80 9.01±0.21 2.92±0.01

Artemia 48.42±1.36 19.20±0.26 7.46±0.21

Fig. 5 a. Excessive protozoan (Zoothamnium sp) infestation on larval body of prawn (Macrobrachium rosenbergii); b. Protozoan growth on empty shells of Artemia; c. Exuvia Entrapment diseases of prawn (Macrobrachium rosenbergii) larvae

16 | InternatIOnal AquAFeed | September-October 2011 September-October 2011 | InternatIOnal AquAFeed | 17

F: Larval diets

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